Actin‐spectrin scaffold supports open fenestrae in liver sinusoidal endothelial cells

Fenestrae are open transmembrane pores that are a structural hallmark of healthy liver sinusoidal endothelial cells (LSECs). Their key role is the transport of solutes and macromolecular complexes between the sinusoidal lumen and the space of Disse. To date, the biochemical nature of the cytoskeleton elements that surround the fenestrae and sieve plates in LSECs remain largely elusive. Herein, we took advantage of the latest developments in atomic force imaging and super‐resolution fluorescence nanoscopy to define the organization of the supramolecular complex(es) that surround the fenestrae. Our data revealed that spectrin, together with actin, lines the inner cell membrane and provided direct structural support to the membrane‐bound pores. We conclusively demonstrated that diamide and iodoacetic acid (IAA) affect fenestrae number by destabilizing the LSEC actin‐spectrin scaffold. Furthermore, IAA induces rapid and repeatable switching between the open vs closed state of the fenestrae, indicating that the spectrin‐actin complex could play an important role in controlling the pore number. Our results suggest that spectrin functions as a key regulator in the structural preservation of the fenestrae, and as such, it might serve as a molecular target for altering transendothelial permeability.     

Endocytosis and degradation of serglycin in liver sinusoidal endothelial cells

We have previously reported that liver sinusoidal endothelial cells (LSECs) are responsible for the clearance of monocyte chondroitin sulfate proteoglycan serglycin from the circulation (øynebråten et al.(2000) J. Leukocyte Biol. 67; 183–188). The aim of the present study was to investigate the kinetics of degradation of endocytosed serglycin in primary cultures of LSECs. The final degradation products of serglycin labelled biosynthetically in the glycosaminoglycan (GAG) chains with [³H] in the acetyl groups of N-acetyl galactosamine residues, [¹⁴C] in the pyranose rings, or [³⁵S] in the sulfate groups were identified as[³H]-acetate, [¹⁴C]-lactate and [³⁵S]-sulfate. Comparison of the rate of release of degradation products from the cells after endocytosis of serglycin labelled chemically with ¹²⁵I in the tyrosine residues, or biosynthetically with [³⁵S] or [³H] in the sulfate or acetyl groups, respectively, showed that ¹²⁵I appeared more rapidly in the medium than [³⁵S]-sulfate and [³H]-acetate. Judging from the speed of appearance of free ¹²⁵I both intracellularly and in the medium, the core protein is degraded considerably more rapidly than the GAG chains.Desulfation of the GAG chains starts after the GAG chains are released from the core protein. Generation of lactate and acetate as the final products from degradation of the carbon skeleton of the GAG chains indicates that catabolism of endocytosed macromolecules in LSECs proceeds anaerobically.     

PeakForce Tapping resolves individual microvilli on living cells

Microvilli are a common structure found on epithelial cells that increase the apical surface thus enhancing the transmembrane transport capacity and also serve as one of the cell's mechanosensors. These structures are composed of microfilaments and cytoplasm, covered by plasma membrane. Epithelial cell function is usually coupled to the density of microvilli and its individual size illustrated by diseases, in which microvilli degradation causes malabsorption and diarrhea. Atomic force microscopy (AFM) has been widely used to study the topography and morphology of living cells. Visualizing soft and flexible structures such as microvilli on the apical surface of a live cell has been very challenging because the native microvilli structures are displaced and deformed by the interaction with the probe. PeakForce Tapping® is an AFM imaging mode, which allows reducing tip–sample interactions in time (microseconds) and controlling force in the low pico‐Newton range. Data acquisition of this mode was optimized by using a newly developed PeakForce QNM‐Live Cell probe, having a short cantilever with a 17‐µm‐long tip that minimizes hydrodynamic effects between the cantilever and the sample surface. In this paper, we have demonstrated for the first time the visualization of the microvilli on living kidney cells with AFM using PeakForce Tapping. The structures observed display a force dependence representing either the whole microvilli or just the tips of the microvilli layer. Together, PeakForce Tapping allows force control in the low pico‐Newton range and enables the visualization of very soft and flexible structures on living cells under physiological conditions. © 2015 The Authors Journal of Molecular Recognition Published by John Wiley & Sons Ltd.     

Lineage tracing reveals conversion of liver sinusoidal endothelial cells into hepatocytes

Although liver sinusoidal endothelial cells (LSECs) have long been known to contribute to liver regeneration following injury, the exact role of these cells in liver regeneration remains poorly understood. In this work, we performed lineage tracing of LSECs in mice carrying Tie2‐Cre or VE‐cadherin‐Cre constructs to facilitate fate‐mapping of LSECs in liver regeneration. Some YFP‐positive LSECs were observed to convert into hepatocytes following a two‐thirds partial hepatectomy (PH). Furthermore, human umbilical vein endothelial cells (HUVECs) could be triggered to convert into cells that closely resembled hepatocytes when cultured with serum from mice that underwent an extended PH. These findings suggest that mature non‐hepatocyte LSECs play an essential role in mammalian liver regeneration by converting to hepatocytes. The conversion of LSECs to hepatocyte‐like (iHep) cells may provide a new approach to tissue engineering.     

Beta-glucuronidase and chloroacetate-esterase staining discriminates rat liver sinusoidal endothelial cells from Kupffer cells in primary culture

Beta-glucuronidase and N-AS-D-chloroacetate esterase cytochemistry have been applied to rat liver sinusoidal endothelial cells and Kupffer cells. Both staining procedures allowed a clear-cut differentiation of either cell type. Kupffer cells which had been stained with beta-glucuronidase showed a positive reaction, whereas sinusoidal endothelial cells were completely negative. If the chloroacetate reaction was used, the former stained diffusely while the latter showed a characteristic granular staining pattern. Identity and purity of sinusoidal endothelial cells and Kupffer cells was validated by transmission and scanning electron microscopy as well as by the pattern of released eicosanoids which is characteristic for either cell type. These two staining techniques are a valuable addition to the peroxidase reaction commonly applied for differentiation.     

BETA-glucuronidase and chloroacetate-esterase staining discriminates rat liver sinusoidal endothelial cells from Kupffer cells in primary culture

Beta-glucuronidase and N-AS-D-chloroacetate esterase cytochemistry have been applied to rat liver sinusoidal endothelial cells and Kupffer cells. Both staining procedures allowed a clear-cut differentiation of either cell type. Kupffer cells which had been stained with beta-glucuronidase showed a positive reaction, whereas sinusoidal endothelial cells were completely negative. If the chloroacetate reaction was used, the former stained diffusely while the latter showed a characteristic granular staining pattern. Identity and purity of sinusoidal endothelial cells and Kupffer cells was validated by transmission and scanning electron microscopy as well as by the pattern of released eicosanoids which is characteristic for either cell type. These two staining techniques are a valuable addition to the peroxidase reaction commonly applied for differentiation.     

Extraction of membrane proteins from a living cell surface using the atomic force microscope and covalent crosslinkers

The force curve mode of the atomic force microscope (AFM) was applied to extract intrinsic membrane proteins from the surface of live cells using AFM tips modified by amino reactive bifunctional covalent crosslinkers. The modified AFM tips were individually brought into brief contact with the living cell surface to form covalent bonds with cell surface molecules. The force curves recorded during the detachment process from the cell surface were often characterized by an extension of a few hundred nanometers followed mostly by a single step jump to the zero force level. Collection and analysis of the final rupture force revealed that the most frequent force values (of the force) were in the range of 0.4–0.6 nN. The observed rupture force most likely represented extraction events of intrinsic membrane proteins from the cell membrane because the rupture force of a covalent crosslinking system was expected to be significantly larger than 1.0 nN, and the separation force of noncovalent ligand-receptor pairs to be less than 0.2 nN, under similar experimental conditions. The transfer of cell surface proteins to the AFM tip was verified by recording characteristic force curves of protein stretching between the AFM tips used on the cell surface and a silicon surface modified with amino reactive bifunctional crosslinkers. This method will be a useful addition to bionanotechnological research for the application of AFM.     

Nanostructure and force spectroscopy analysis of human peripheral blood CD4+ T cells using atomic force microscopy

To date, nanoscale imaging of the morphological changes and adhesion force of CD4⁺ T cells during in vitro activation remains largely unreported. In this study, we used atomic force microscopy (AFM) to study the morphological changes and specific binding forces in resting and activated human peripheral blood CD4⁺ T cells. The AFM images revealed that the volume of activated CD4⁺ T cells increased and the ultrastructure of these cells also became complex. Using a functionalized AFM tip, the strength of the specific binding force of the CD4 antigen-antibody interaction was found to be approximately three times that of the unspecific force. The adhesion forces were not randomly distributed over the surface of a single activated CD4⁺ T cell, indicated that the CD4 molecules concentrated into nanodomains. The magnitude of the adhesion force of the CD4 antigen-antibody interaction did not change markedly with the activation time. Multiple bonds involved in the CD4 antigen-antibody interaction were measured at different activation times. These results suggest that the adhesion force involved in the CD4 antigen-antibody interaction is highly selective and of high affinity.     

Liver sinusoidal endothelial cells orchestrate NK cell recruitment and activation in acute inflammatory liver injury

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Atomic force microscopy comes of age

AFM (atomic force microscopy) analysis, both of fixed cells, and live cells in physiological environments, is set to offer a step change in the research of cellular function. With the ability to map cell topography and morphology, provide structural details of surface proteins and their expression patterns and to detect pico‐Newton force interactions, AFM represents an exciting addition to the arsenal of the cell biologist. With the explosion of new applications, and the advent of combined instrumentation such as AFM—confocal systems, the biological application of AFM has come of age. The use of AFM in the area of biomedical research has been proposed for some time, and is one where a significant impact could be made. Fixed cell analysis provides qualitative and quantitative subcellular and surface data capable of revealing new biomarkers in medical pathologies. Image height and contrast, surface roughness, fractal, volume and force analysis provide a platform for the multiparameter analysis of cell and protein functions. Here, we review the current status of AFM in the field and discuss the important contribution AFM is poised to make in the understanding of biological systems.     

Long-term preservation of high endocytic activity in primary cultures of pig liver sinusoidal endothelial cells

Together with Kupffer cells, liver sinusoidal endothelial cells (LSECs) constitute the most powerful scavenger system in the body. However, studies on LSEC function are hampered by the fact that the cells lose their scavenger ability and start deteriorating after a few days in culture. The purpose of the present study was to improve the conditions of cultivation to prolong the survival of pig LSECs in vitro. We used the high capacity receptor-mediated endocytosis of soluble waste molecules as a marker for functionally intact cells in the cultures. Compared with two commercially-, and two other media specifically designed for use with either SECs or hepatocytes from rat, our newly developed serum-free medium, DM 110/SS, devoid of any components of animal origin, was superior in maintaining the endocytic activity. Of six growth factors studied for their effect on endocytosis, basic fibroblast, and recombinant epidermal, but not vascular endothelial growth factor, were found to be most beneficial. After 8 days in DM 110/SS, LSECs maintained endocytosis via the scavenger receptor, mannose receptor, collagen alpha-chain receptor and the Fc-gamma receptor. All endocytosed ligands, except for aggregated IgG were degraded in 8-day-old cultures. Using the new medium, the cells endocytosed ligands for up to 20 days, and survived for at least an additional 10 days, albeit without the high endocytic activity typical of intact LSECs. Importantly, DNA synthesis in prolonged cultures of LSECs was observed only when maintained in DM 110/SS medium. In conclusion, we describe a protocol for the maintenance of LSECs in culture for the longest period yet reported.     

Culture and characterization of sinusoidal endothelial cells isolated from human liver

Summary Although most vascular models use large vessel endothelial cells from human umbilical veins, there is marked heterogeneity among endothelial cells from different vascular beds and organs. More accurate modeling of endothelial involvement in liver diseases, including metastasis, may result from the use of human hepatic sinusoidal endothelial cells. Liver resection specimens were sectioned, then treated with a 1.2 U/ml dispase solution. The tissue slurry was mechanically disaggregated and separated by centrifugation on a Percoll density gradient. Cells were then cultured in an endothelial-specific media with growth factors. These techniques resulted in a homogeneous monolayer consistent with endothelial cells by light microscopy. An endothelial origin was further confirmed by the expression of Factor VIII, binding of Ulex lectin, and uptake of acetylated low density lipoprotein. Electron microscopy showed transcellular fenestrations consistent with a sinusoidal origin. These human hepatic sinusoidal endothelial cells were then studied for expression of the adhesion molecules CD31/PECAM, CD34, E-selectin, ICAM-1, L-selectin, LFA-3, P-selectin, and VCAM-1 plus the binding of wheat germ agglutinin lectin. The patterns of adhesion molecule expression and lectin binding by these cells are characteristic of hepatic sinusoidal endothelia. In this paper, we have described a method for isolation and culture of human cells with the morphologic and phenotypic characteristics of hepatic sinusoidal endothelia.     

应用原子力显微镜分析正常淋巴细胞和Jurkat细胞的形态和机械性质

淋巴细胞形态和机械性质的变化与人的健康、疾病的治疗和诊断有着密切关系。本研究利用原子力显微镜研究淋巴细胞和Jurkat细胞形态和机械性质。结果显示,这2种细胞的形态较为相似,但通过对力曲线的分析得出这2种细胞的机械性质明显不同。正常淋巴细胞粘弹力范围大致为(796.7±248.5)pN,而Jurkat细胞分布于(158.5±37.5)pN;正常淋巴细胞的杨氏模量(0.471kPa±0.081kPa)近4倍于Jurkat细胞(0.0964kPa±0.0229kPa);而Jurkat细胞(4.322mN/m±0.382mN/m)的硬度近2倍于正常淋巴细胞(2.278mN/m±0.488mN/m)。结果表明原子力显微镜能可在临床诊断上区分正常细胞与肿瘤细胞,即使两者形态区别不明显。     

Abscisic acid does not evoke calcium influx in murine primary microglia and immortalised murine microglial BV-2 and N9 cells

Brain microglia are resident macrophage-like cells representing the first and main form of active immune response during brain injury. Microglia-mediated inflammatory events in the brain are known to be associated with chronic degenerative diseases such as Multiple Sclerosis, Parkinson’s, or Alzheimer’s disease. Therefore, identification of mechanisms activating microglia is not only important in the understanding of microglia-mediated brain pathologies, but may also lead to the development of new anti-inflammatory drugs for the treatment of chronic neurodegenerative diseases. Recently, abscisic acid (ABA), a phytohormone regulating important physiological functions in higher plants, has been proposed to activate murine microglial cell line N9 through increased intracellular calcium. In the present study, we determined the response to ABA and its analogues from murine primary microglia and immortalized murine microglial cell line BV-2 and N9 cells. A Fura-2-acetoxymethyl ester (Fura-2AM)-based ratiometric calcium imaging and measurement technique was used to determine the intracellular calcium changes in these cells when treated with (−)-ABA, (+)-ABA, (−)-trans-ABA and (+)-trans-ABA. Both primary microglia and microglial cell lines (BV-2 and N9 cells) showed significant increase in intracellular calcium ([Ca²⁺]i) in response to treatment with ATP and ionomycine. However, ABAs failed to evoke dose- and time-dependent [Ca²⁺]i changes in mouse primary microglia, BV-2 and N9 cells. Together, these surprising findings demonstrate that, contrary to that reported in N9 cells [3], ABAs do not evoke intracellular calcium changes in primary microglia and microglial cell lines. The broad conclusion that ABA evokes [Ca²⁺]i in microglia requires more evidence and further careful examination.     

Raman spectroscopy–based insight into lipid droplets presence and contents in liver sinusoidal endothelial cells and hepatocytes

Liver sinusoidal endothelial cells (LSECs), a type of endothelial cells with unique morphology and function, play an important role in the liver hemostasis, and LSECs dysfunction is involved in the development of nonalcoholic fatty liver disease (NAFLD). Here, we employed Raman imaging and chemometric data analysis in order to characterize the presence of lipid droplets (LDs) and their lipid content in primary murine LSECs, in comparison with hepatocytes, isolated from mice on high‐fat diet. On NAFLD development, LDs content in LSECs changed toward more unsaturated lipids, and this response was associated with an increased expression of stearylo‐CoA desaturase‐1. To the best of our knowledge, this is a first report characterizing LDs in LSECs, where their chemical composition is analyzed along the progression of NAFLD at the level of single LD using Raman imaging.      

Direct mapping of melanoma cell ‐ endothelial cell interactions

The most life‐threatening aspect of cancer is metastasis; cancer patient mortality is mainly due to metastasis. Among all metastases, presence of brain metastasis is one with the poorest prognosis; the median survival time can be counted in months. Therefore, prevention or decreasing their incidence would be highly desired both by patients and physicians. Metastatic cells invading the brain must breach the cerebral vasculature, primarily the blood‐brain barrier. The key step in this process is the establishment of firm adhesion between the cancer cell and the cerebral endothelial layer. Using the atomic force microscope, a high‐resolution force spectrograph, our aim was to explore the connections among the cell morphology, cellular mechanics, and biological function in the process of transendothelial migration of metastatic cancer cells. By immobilization of a melanoma cell to an atomic force microscope's cantilever, intercellular adhesion was directly measured at quasi‐physiological conditions. Hereby, we present our latest results by using this melanoma‐decorated probe. Binding characteristics to a confluent layer of brain endothelial cells was directly measured by means of single‐cell force spectroscopy. Adhesion dynamics and strength were characterized, and we present data about spatial distribution of elasticity and detachment strength. These results highlight the importance of cellular mechanics in brain metastasis formation and emphasize the enormous potential toward exploration of intercellular dynamic‐related processes.     

Nitric oxide decreases the sensitivity of pulmonary endothelial cells to LPS-induced apoptosis in a zinc-dependent fashion

We hypothesized that: (a) S-nitrosylation of metallothionein (MT) is a component of pulmonary endothelial cell nitric oxide (NO) signaling that is associated with an increase in labile zinc; and (b) NO mediated increases in labile zinc in turn reduce the sensitivity of pulmonary endothelium to LPS-induced apoptosis. We used microspectrofluorometric techniques to show that exposing mouse lung endothelial cells (MLEC) to the NO-donor, S-nitrosocysteine, resulted in a 45% increase in fluorescence of the Zn²⁺-specific fluorophore, Zinquin, that was rapidly reversed by exposure to the Zn²⁺ chelator, NNNN-tetrakis-(2-pyridylmethyl)ethylenediamine; TPEN). The absence of a NO-mediated increase in labile Zn²⁺ in MLEC from MT-I and -II knockout mice inferred a critical role for MT in the regulation of Zn²⁺ homeostasis by NO. Furthermore, we found that prior exposure of cultured endothelial cells from sheep pulmonary artery (SPAEC), to the NO-donor, S-nitroso-N-acetylpenicillamine (SNAP) reduced their sensitivity to lipopolysaccharide (LPS) induced apoptosis. The anti-apoptotic effects of NO were significantly inhibited by Zn²⁺ chelation with low doses of TPEN (10 M). Collectively, these data suggest that S-nitrosylation of MT is associated with an increase in labile (TPEN chelatable) zinc and NO-mediated MT dependent zinc release is associated with reduced sensitivity to LPS-induced apoptosis in pulmonary endothelium.     

Receptor-mediated endocytosis of aldehyde-modified proteins by sinusoidal liver cells

Formaldehyde-treated serum albumin (f-Alb) is known to be endocytosed by sinusoidal lever cells via a receptor-mediated mechanism. The receptor purified from rat livers exhibited a molecular weight of 125,000, consisting of two glycoprotein components with molecular weights of 53,000 and 30,000, respectively. Experiments using antireceptor antibody demonstrated that the f-Alb receptor is distinct from the receptor that mediates endocytotic uptake of acetylated low-density lipoprotein, but they share a common property of being inhibited by several polyanions, suggesting that polyanion-sensitivity might play an important role in the scavenger function of simusoidal liver cells. Studies on the ligand specificity of this receptor revealed that a covalent modification by formaldehyde of a limited number of lysine residues in albumin has led to the formation of a receptor-recognition domain(s). Furthermore, in addition to formaldehyde, the ligand activity was also generated with albumin modified by other aliphatic aldehydes, such as glycoaldehyde and glyceraldehyde. This phenomenon was extended to several proteins other than albumin. These data suggest therefore that the f-Alb receptor originally described as being specific for albumin modified by formaldehyde may play a general role as a scavenger receptor for aldehyde-modified proteins.This article was presented during the proceedings of the International Conference on Macromolecular Structure and Function, held at the National Defence Medical College, Tokorozawa, Japan, December 1985.     

Structure and dynamics of the fusion pore in live cells.

Atomic force microscopy reveal pit-like structures typically containing three or four, approximately 150 nm in diameter depressions at the apical plasma membrane in live pancreatic acinar cells. Stimulation of secretion causes these depressions to dilate and return to their resting size following completion of the process. Exposure of acinar cells to cytochalasin B results in decreased depression size and a loss in stimulable secretion. It is hypothesized that depressions are the fusion pores, where membrane-bound secretory vesicles dock and fuse to release vesicular contents. Zymogen granules, the membrane-bound secretory vesicles in exocrine pancreas, contain the starch digesting enzyme, amylase. Using amylase-specific immunogold labeling, localization of amylase at depressions following stimulation of secretion is demonstrated. This study confirms depressions to be the fusion pores in pancreatic acinar cells. High-resolution images of the fusion pore in live pancreatic acinar cells reveal the structure in much greater detail than has previously been observed.